Stevioside, a major constituent of Stevia rebaudiana Bertoni, is commonly used as a sugar substitute in Japan. Introduction within the U.S. is currently being considered. A structurally related constituent of S. rebaudiana, rebaudioside A, also has strong economic potential as a commercial sweetening agent due to its pleasurable taste characteristics. Utilization of sucsucrose substitutes is highly desirable. A question of great importance, however, is the relative safety with which such substances can be ingested by humans. In essence, the use of stevioside in Japan can be considered and extensive clinical trial. No human toxicity has thus far been reported, and this is consistent with the lack of activity demonstrated in various biological assays. We have confirmed reports that stevioside is not a bacterial mutagen. Similarly, rebaudioside A was found to be inactive. Importantly, however, the aglycone of stevioside and rebaudioside A, steviol, is highly mutagenic in the presence of a metabolic activating system. Steviol 16 Beta, 17 Beta-oxide was identified as the putative active metabolite. Presently, we propose to provide additional characterization of the potential of stevioside to mediate mutagenicity. The enzymic requirements involved in the metabolic conversion of steviol, and related entkaurene derivatives, will be studied in detail. Additionally, the ability of purified epoxide hydrolase to detoxify the active mutagenic metabolite(s) will be elucidated. As covalent interaction with DNA is clearly related to mutagenesis, this will also be assessed. Further, through evaluation of natural or synthetic congeners, the steps of hydrolytic cleavage required to yield a stevioside species capable of causing mutation will be determined. Lastly, evaluations will be performed with cultured C3H/10T 1/2 cells to provide a relevant indication of carcinogenic potential. In addition to the heuristic value of characterizing potential sweetening agents by the procedures described herein, academic value of characterizing potential sweetening agents by the procedures described herein, academic value stems from exploring ent-kaurene-mediated mutagenicity. Perhaps more importantly, the studies will be of value in determining the future worldwide utilization of stevioside and rebaudioside A.